The development of gene therapy for hemophilia and other diseases remains stymied by a failure to achieve clinically significant levels of transgene integration in targeted cells. Adeno-associated virus integrates into AAVS1, a site on Human Chromosome 19qter (AAVS1). Increasing evidence indicates the involvement of both the viral Rep protein and the AAV Inverted Terminal Repeats (ITR) in this integration process. The goal of this project is to utilize AAV functions in order to develop non-viral vectors that will result in the targeted integration of heterologous transgenes, including the genes for Human Coagulation Factors VIII and IX. Results: (1) In non-clonal Hepatoma G-2 cells, codelivery of the Rep protein by lipid-based transfection resulted in a long-term, Rep dose-dependent increase in the expression of a marker gene flanked by AAV ITRs; (2) In 293 cells the presence of both Rep (delivered by liposomes as either a protein or as a gene) and AAV ITRs increased the frequency of clonal cells with longterm transgene expression. (3) In order to create potentially useful in vitro and in vivo models of AAV integration, we developed murine cell lines and animals transgenic for AAVS1. These models are currently being evaluated using non-viral AAV vectors for Human Coagulation Factor IX.